Home › Cell Biology › In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
Cell Biology JoVE (Open Access) Citable · DOI

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

DOI: 10.3791/4417-v
What you'll learn
  • ✓ Synthesize three distinct collagen fibril architectures from Type I monomer
  • ✓ Manipulate assembly conditions to control collagen structural morphology
  • ✓ Characterize collagen fibrils using atomic force microscopy
  • ✓ Verify fibril type and quality through AFM imaging analysis
Protocol

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Difficulty
intermediate
Total time
~4–6 hours per collagen type (assembly + AFM characterization)
Biosafety
BSL-1

Steps

1
Prepare native collagen fibrils

Assemble Type I collagen monomers under physiological conditions to form native-state fibrous collagen. This baseline assembly serves as a structural reference.

▶ 01:06
2
Prepare fibrous long spacing collagen

Alter assembly conditions (pH, salt, or temperature) to direct collagen monomers into fibrous long spacing morphology with characteristic quasi-crystalline banding.

▶ 01:45
3
Prepare segmental long spacing collagen

Expose collagen monomers to distinct chemical or physical conditions to generate segmental long spacing architecture, a third structurally distinct assembly type.

▶ 02:35
4
Characterize collagen fibrils by atomic force microscopy

Prepare samples and acquire high-resolution AFM images of each collagen assembly to visualize nanoscale fibril morphology and banding patterns.

▶ 03:19
5
Verify collagen type and fibril quality

Analyze AFM results to confirm successful synthesis of native, fibrous long spacing, and segmental long spacing collagen based on characteristic ultrastructural signatures.

▶ 05:10
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